Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos.

Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus-oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

Expression levels of FSHR, IGF1R, CYP11al and HSD3ß in cumulus cells can predict in vitro developmental competence of bovine oocytes.

The efficiency of in vitro embryo production technologies would be improved by the development of suitable non-invasive biomarkers that allow the selection of good quality cumulus-oocyte complexes (COCs). The present study used whole, single oocyte culture to investigate whether the expression levels of follicle-stimulating hormone receptor (FSHR), insulin-like factor 1 receptor (IGF1R) and three steroidogenesis-related enzymes (CYP11al, CYP19al and HSD3ß) in cumulus cells reflected the developmental competence of COCs. Cumulus cells were collected from single COCs before maturation culture and relative mRNA levels were assessed using real-time PCR. The analysis indicated that mRNAs for FSHR, IGF1R, CYP11al and HSD3ß were present at higher levels in cumulus cells from COCs that failed to form blastocysts compared with cumulus cells from COCs that formed blastocysts. Moreover, FSHR and IGF1R mRNA levels were positively correlated with those of genes for steroidogenesis-related enzymes. In conclusion, poor developmental competence of COCs was related to higher expression of FSHR, IGF1R, CYP11al and HSD3ß in cumulus cells, which may indicate the advanced differentiation of cumulus cells into granulosa cells.

Effects of a tunnel ventilation system within the tie-stall barn environment upon the productivity of dairy cattle during the winter season.

OBJECTIVE: The objective of this study was to examine the effect of using a tunnel ventilation system within the dairy barn environment upon the productivity of dairy cows during the winter season. METHODS: The study was performed at the University Farm, Faculty of Agriculture, Utsunomiya University. Twenty-one Holstein dairy cows (5 heifers and 16 multiparous) were enclosed in a stall barn. Unventilated (UV) and tunnel-ventilated (TV) was operated by turns every other week, and a number of key parameters were measured in the barn, including tunnel ventilation output, temperature, relative humidity, gas concentrations (oxygen [O2], carbon dioxide [CO2], and ammonia [NH3]). Also, skin and rectal temperature, respiratory rate, blood gas concentrations, and bacterial count were measured from nipple attachments on ten cows. The amount of fodder left uneaten, and general components and somatic cell count of the milk were measured. RESULTS: As for our dairy barn environment, air temperature dropped significantly with the passage of time with TV. Humidity was significantly higher with TV at 0600 h compared to UV, while CO2 and NH3 concentrations with UV were significantly higher than with TV at 0000 h and 0600 h. Skin temperature was significantly lower with TV compared to UV at 0000 h and 0600 h. Respiratory rate was also significantly lower at 0600 h with TV than with UV. Bacterial count for the nipple attachments was significantly lower with TV than with UV at 0600 h. The amount of leftover fodder was significantly less with TV in comparison with UV. CONCLUSION: Our results suggest that a TV system in the winter barn results in environmental improvements, such as reductions in unfavorable gas concentrations and bacterial growth. Consequently, it is expected that barns utilizing a TV system will be beneficial for both animal health and production.

Effects of insulin-like growth factor-1 on the in vitro maturation of canine oocytes.

The maturation rate of canine oocytes during in vitro maturation (IVM) needs to be improved. The present study was designed to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the IVM of canine oocytes. Ovaries were obtained by ovariohysterectomy and were sliced to release cumulus-oocyte complexes (COCs). In Experiment 1, the effects of different concentrations of IGF-1 on the nuclear maturation of oocytes was investigated. The COCs were cultured in a modified medium (mTCM199) with IGF-1 (0, 0.5, 5, 10, and 50 µg/ml). At the end of the 48 h culture, oocytes were fixed and stained to evaluate their nuclear stage. Supplementation with 50 µg/ml IGF-1 induced a significantly higher metaphase II (MII) rate (P < 0.05) compared to the 0 and 0.5 µg/ml IGF-1 groups. In Experiment 2, the expression levels of insulin receptor (INSR), IGF-1 receptor (IGF-1R), and IGF-2 receptor (IGF-2R) genes, localized to canine oocytes and cumulus cells, were investigated before and after IVM. The expression level of IGF-1R in cumulus cells after IVM was higher than that before IVM (P < 0.05). In Experiment 3, it was investigated whether an inhibitor of PTEN (phosphatase and tensin homolog), bpV, affects the nuclear maturation of oocytes. Regardless of bpV supplementation at a concentration of 0.2 to 200 µmol/l, there was no significant difference in the proportion of oocytes that reached the MII stage. These results indicated that IGF-1 has a favorable effect on the IVM of canine oocytes, possibly through the stimulation of the Ras/MAPK pathway via IGF-1R expressed in cumulus cells.

Influence of maternal remifentanil concentration on fetal-to-maternal ratio in pregnant ewes.

PURPOSE: Maternal remifentanil infusion is used for minimally invasive fetal surgery or ex-utero intrapartum treatment. The fetal-to-maternal (F/M) ratio of remifentanil concentration at various dosing regimens is useful to manage remifentanil effects. The aim of this study was to investigate the F/M ratio of remifentanil at various concentrations. METHODS: Five pregnant ewes received continuous remifentanil infusion under propofol anesthesia. The remifentanil infusion rate was increased by 0.4 µg/kg/min every 15 min. The response to tail clamping in fetuses was assessed immediately before the change of infusion rate. Arterial remifentanil concentrations in the mother and fetus were determined at each tail clamp. After observing a loss of response to tail clamping, remifentanil infusion was terminated and the concentrations were assessed. RESULTS: The median remifentanil maximum infusion rate and maternal concentration were 3.0 µg/kg/min (range 2.4-3.6) and 21.6 (range 18.0-29.9) ng/mL, respectively. During continuous infusion, the F/M ratio was 0.15 (0.07-0.17), and the slope of the linear regression for the F/M ratio versus infusion rate in each individual was -0.001 ± 0.012/µg kg min (P = 0.876 vs hypothetical value of 0). The F/M ratio at the first sampling point in the elimination phase [0.33 (0.07-0.65)] was higher (P = 0.033) than at the last sampling point during continuous infusion [0.15 (0.06-0.17)]. CONCLUSION: The F/M ratio was constant at a steady state regardless of the remifentanil concentration up to 29.9 ng/mL, and increased in the elimination phase in pregnant ewes.

Deficiencies in extrusion of the second polar body due to high calcium concentrations during in vitro fertilization in inbred C3H/He mice.

Successful in vitro fertilization (IVF) of all inbred strains of laboratory mice has not yet been accomplished. We have previously shown that a high calcium concentration improved IVF in various inbred mice. However, we also found that in cumulus-free ova of C3H/He mice such IVF conditions significantly increased the deficiency of extrusion of the second polar body (PBII) in a dose-dependent manner (2% at 1.71 mM and 29% at 6.84 mM, P < 0.05) and that PBII extrusion was affected by high calcium levels at 2-3 h post-insemination. While developmental competence of ova without PBII extrusion to blastocysts after 96 h culture was not affected, a significant reduction in the nuclear number of the inner cell mass was observed in blastocyst fertilized under high calcium condition. We also examined how high calcium concentration during IVF affects PBII extrusion in C3H/He mice. Cumulus cells cultured under high calcium conditions showed a significantly alleviated deficient PBII extrusion. This phenomenon is likely to be specific to C3H/He ova because deficient PBII extrusion in reciprocal fertilization between C3H and BDF1 gametes was observed only in C3H/He ova. Sperm factor(s) was still involved in deficient PBII extrusion due to high calcium concentrations, as this phenomenon was not observed in ova activated by ethanol. The cytoskeletal organization of ova without PBII extrusion showed disturbed spindle rotation, incomplete formation of contractile ring and disturbed localization of actin, suggesting that high calcium levels affect the anchoring machinery of the meiotic spindle. These results indicate that in C3H/He mice high calcium levels induce abnormal fertilization, i.e. deficient PBII extrusion by affecting the cytoskeletal organization, resulting in disturbed cytokinesis during the second meiotic division. Thus, use of high calcium media for IVF should be avoided for this strain.

Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination.

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.

Advancements in medical research using fetal sheep: Implications for human health and treatment methods.

Sheep are typically considered as industrial animals that provide wool and meals. However, they play a significant role in medical research in addition to their conventional use. Notably, sheep fetuses are resistant to surgical invasions and can endure numerous manipulations, such as needle puncture and cell transplantation, and surgical operations requiring exposure beyond the uterus. Based on these distinguishing characteristics, we established a chimeric sheep model capable of producing human/monkey pluripotent cell-derived blood cells via the fetal liver. Furthermore, sheep have become crucial as human fetal models, acting as platforms for developing and improving techniques for intrauterine surgery to address congenital disorders and clarifying the complex pharmacokinetic interactions between mothers and their fetuses. This study emphasizes the significant contributions of fetal sheep to advancing human disease understanding and treatment strategies, highlighting their unique characteristics that are not present in other animals.

The developmental potential of oocytes is impaired in cattle with liver abnormalities.

Here, we investigated the effect of liver abnormality on the developmental potential of bovine oocytes. Good quality oocytes from healthy cows and from animals with a liver abnormality were matured and fertilized in vitro and then cultured to the blastocyst stage. On day 7 after fertilization, embryo cleavage and development were assessed. The concentrations of glucose, nonesterified fatty acid (NEFA), γ-glutamyl transpeptidase (γ-GTP), ß-hydroxybutyrate (BHBA) and glutathione were measured in follicular fluids (FF). The proportion of good quality oocytes and the frequency of development to the blastocyst stage were lower in the liver anomaly group than those of the control group (P<0.05). The concentrations of γ-GTP and BHBA in the FF of the liver anomaly group were higher than those of the control group (P<0.05). The concentration of glutathione in the FF of the liver anomaly group was lower than that of the control group (P<0.05). Moreover, there was a negative correlation between these concentrations and the proportions of oocytes that developed to the blastocyst stage (P<0.05). Supplementation of the culture medium with γ-GTP or BHBA did not affect the rate of oocyte maturation but did cause a concentration-dependent reduction in the frequency of fertilized oocytes that developed to the blastocyst stage. Our findings indicate that the quality of oocytes and their potential for development are lower in cattle with liver disorders than those in healthy cattle; one possible cause may be the high concentration of γ-GTP and/or BHBA in their FF.

Effect of polyvinylpyrrolidone on sperm function and early embryonic development following intracytoplasmic sperm injection in human assisted reproduction.

The objective here was to review the effects of polyvinylpyrrolidone (PVP) upon sperm function and embryonic development in humans. PVP has been used successfully in intracytoplasmic sperm injection (ICSI) to facilitate the handling and immobilization of sperm for both domestic animals and humans. In our previous reports, PVP solution exists locally in embryos injected during the early developmental period, and also exerts influence over the developmental capacity of such embryos. In other reports, PVP causes significant damage to sperm membranes that can be detected by transmission electron microscopy, and has been associated with chromosomal abnormalities in pregnancy derived from ICSI embryos. In some Japanese clinics, PVP-free media has been used for sperm immobilization in order to optimise safety. Consequently, it is strongly suggested that the success rate of fertilization and clinical pregnancy could be improved by using PVP-free solution for human ICSI. In conclusion, our interpretation of the available data is to perform ICSI without PVP or select a lower concentration of PVP solution in order to reduce safety for pregnancy and children born via ICSI.

The secretion and metabolism of cumulus cells support fertilization in the bovine model.

The cumulus oophorus is a structure that surrounds the mammalian egg and plays a key role in fertilization. However, very little is known with regards to how secretions from the cumulus cells can specifically promote fertilization. We hypothesized that secretions from bovine cumulus cells, and the reduction of oxygen stress by metabolic change, would enhance the fertilization capacity of sperm during in vitro fertilization (IVF) procedures. To prove our hypothesis, sperm were pre-incubated in chemically defined capacitation media containing methyl-beta-cyclodextrin and used to inseminate cumulus cell oocyte complexes, or denuded oocytes, with some components. While sperm capacitation was induced in capacitation media, fertilization was impeded by the removal of cumulus cells from cumulus cell oocyte complexes. Secretions from cumulus cells promoted the formation of two pronuclei via a filter and the fertilization of denuded oocytes was dramatically enhanced with hyaluronate, low oxygen concentration, or progesterone in fertilization media (P < 0.05). This demonstrates that these factors-maintained sperm motility and capacitation or enhanced the hyper-activation of capacitated sperm (P < 0.05). We conclude that cumulus cells secrete progesterone, hyaluronate and undergo metabolic events to reduce oxidative stress in fertilization media. These phenomenons help to improve the fertilization capacity of sperm. We believe that this study makes a significant contribution to our understanding of the function of cumulus cells during fertilization in animals and humans.

Capacitation status of activated bovine sperm cultured in media containing methyl-ß-cyclodextrin affects the acrosome reaction and fertility.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. One of the key processes involved in capacitation is the activation of sperm motility. Here, we investigated the capacitation and fertility status of activated sperm which had been cultured in media containing methyl-ß-cyclodextrin (MBCD). In order to do this, single activated sperm were caught using a micropipette and stained with chlortetracycline (CTC). Firstly, we investigated the effects of preincubation upon motility, capacitation of activated sperm and fertility. Culture in preincubation media supplemented with MBCD increased the rates of activation and fertilization compared with sperm cultured by control methods (p < 0.05). Following capture, individual activated sperm mostly exhibited a pattern characteristic of capacitation.Secondly we examined the effects of culturing sperm in media with or without glucose (G) and pyruvate acid (P) upon activated motility, the capacitation of activated sperm and fertility. Supplementation of culture media with G and P resulted in higher proportions of activated sperm and increased fertilization rates compared to culture without G and P (p < 0.05). Most of the sperm activated by culture in G and P exhibited patterns characteristic of capacitation. Without G and P, individual activated sperm mostly exhibited patterns characteristic of the acrosome reaction (p < 0.05). In conclusion, activated sperm exhibited patterns characteristic of capacitation. In addition, sperm activated in media containing an energy source (glucose and pyruvate acid) appeared to exhibit acrosome reactiveness and fertility.

The effects of glutathione ethyl ester in in vitro maturation on the developmental ability of oocytes derived from cattle with liver abnormalities.

The main objectives of this study was to identify the effects of a relationship of hyper-concentration of Gamma-glutamyltransferase (γ-GTP) in follicle fluid (FF) on the levels of glutathione (GSH)/reactive oxygen species (ROS) in oocytes and subsequent embryo development in cattle with abnormal livers. Furthermore, we investigated the effect of supplementing in vitro maturation medium with glutathione ethyl ester (GSH-OEt) on the subsequent developmental potential of oocytes from such cattle. We used a control group of cattle (with normal livers) and a liver disorder (LD) group, in which the liver was diagnosed as being abnormal. In experiment 1, the LD group was divided to two subgroups according to the concentration of γ-GTP in FF: a low group (≤50 IU/L; the low LD group), and a high group (>50 IU/L: the high LD group). Cumulus oocyte-complexes (COCs) were matured and fertilized in vitro and then cultured to the blastocyst stage. The levels of GSH and ROS in the matured oocytes after IVM were then assessed in each group. On day 7 after fertilization, embryo cleavage and development were assessed. We found that the rate of development to the blastocyst stage was significantly lower in the high LD group than in the control group and the low LD group. The levels of GSH in matured oocytes were significantly lower in the high LD group than in the control group and low LD group. The levels of ROS in matured oocytes was significantly higher in the high LD group than in the control group and the low LD group. In experiment 2, COCs from cattle in the high LD group were matured in m-199 supplemented with 5 mM GSH-OEt, then IVF and IVC was performed for 7 days. The GSH levels were determined in some COCs after IVM. The supplementation of media with GSH-OEt during IVM increased the levels of GSH in mature oocytes and improved the rate of blastocyst development compared with the control group. In conclusion, GSH-OEt supplementation to media during IVM can improve the developmental potential of oocytes in liver-diseased cattle with high γ-GTP concentrations in the FF by increasing intracellular GSH synthesis and scavenging ROS.

Fetal sheep support the development of hematopoietic cells in vivo from human induced pluripotent stem cells.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

The effects of methyl-ß-cyclodextrin on in vitro fertilization and the subsequent development of bovine oocytes.

In this study, we investigated the effects of methyl-ß-cyclodextrin (MBCD) on in vitro fertilization and the subsequent development of bovine oocytes. Bovine oocytes matured in serum-free medium were inseminated with frozen-thawed sperm pre-incubated in protein-free modified Brackett and Oliphant medium (BO) containing various concentrations of MBCD for various periods. MBCD decreased the frequency of live sperm, however enhanced the capacitation and acrosome reaction of the live sperm. Pre-incubation of sperm with 0.5, 1.0 and 1.5 mM MBCD for 2 and 4 h increased the frequency of normal fertilization. Embryos derived from oocytes fertilized with spermatozoa pre-incubated with MBCD developed normally to the blastocyst stage and term. There were individual differences and similar tendencies in four different sires in terms of the effects of MBCD upon fertilization. These results indicate that the pre-incubation of bovine sperm with MBCD affects viability and capacitation status of the sperm and promotes fertilization in vitro. Embryos derived from oocytes fertilized with sperm pre-incubated with MBCD developed normally to the blastocyst stage and term.

Antioxidant treatment during preservation of bovine ovaries increased the development potential of embryos.

The overnight preservation of bovine ovaries would be highly useful in the subsequent harvest of viable oocytes for reproductive study. The present study aimed to optimize conditions for overnight preservation of bovine ovaries by examining the effects of temperature, solution and supplementation. In Experiment 1, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved at 15°C was higher than that at either 5 or 25°C (p < 0.05). In Experiment 2, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved in University of Wisconsin solution was higher than when PBS or saline was used (p < 0.05). In Experiment 3, oocytes preserved in saline supplemented with 0.3 mM glutathione (GSH) exhibited an increase in the rate of blastocyst formation compared with oocytes supplemented with 0 or 3 mM GSH (p < 0.05). In Experiment 4, supplementation with 10 µM epigallocatechin gallate during ovary preservation increased the rate of blastocyst formation (p < 0.05). The blastocysts derived from ovaries stored in saline supplemented with GSH at 15°C for 24 h were shown to develop into normal offsprings following transfer to recipient heifers. Our studies indicate that bovine IVM/IVF embryos derived from ovaries preserved in saline supplemented with an antioxidant at 15°C for 24 h can successfully develop to the blastocyst stage and result in offspring.

Detrimental effects of oxidative stress in bovine oocytes during intracytoplasmic sperm injection (ICSI).

Intracytoplasmic sperm injection (ICSI) is an essential technology in animal and human reproduction. However, the developmental competence and pregnancy rate of embryos derived from ICSI are still lower than that from the conventional in vitro fertilization technique. In this report, we focused on reactive oxygen species (ROS) as a potential detrimental factor for ICSI. Experiment 1 was conducted to evaluate the effect of oxidative stress by two different oxygen concentrations (20%: control vs. 5%) in ICSI on the developmental competence (blastocyst rate: day 7, DNA fragmentation rate: day 4) and, ROS concentration and mitochondrial membrane potential of oocytes in ICSI. In the 5% O2 group, the blastocyst rate (29.5%) was higher and DNA fragmentation rate (4.8 ±â€¯1.0%) was lower than those in the control group significantly (12.7% and 18.2 ±â€¯2.4%, respectively, P < 0.05). Also, ROS concentration in the 5% O2 group (12.8 ±â€¯0.7) was significantly lower than that in the control group (47.8 ±â€¯6.9, P < 0.05). In experiment 2, we examined the supplementation of media with reduced glutathione (GSH) during ICSI procedure in an attempt to reduce the oxidative stress. The addition of GSH to the culture medium improved the blastocyst rate (17.6% vs. 30.4%, P < 0.05), and decreased the ROS levels in the oocytes (70.0 ±â€¯7.4 vs. 23.9 ±â€¯4.0, P < 0.05). In conclusion, our present study revealed that oocytes are under oxidative stress in ICSI procedure. Reduction of the oxygen concentration to 5% in the culture environment, or the addition of GSH in to the medium during ICSI procedure can promote the normal embryo development following the ICSI.

Sustained macroscopic engraftment of cynomolgus embryonic stem cells in xenogeneic large animals after in utero transplantation.

Because embryonic stem (ES) cells are able to proliferate indefinitely and differentiate into any type of cell, they have the potential for providing an inexhaustible supply of transplantable cells or tissues. However, methods for the in vitro differentiation of human ES cells are still quite limited. One possible strategy would be to generate differentiated cells in vivo. In view of future clinical application, we investigated the possibility of using xenogeneic large animals for this purpose. We transplanted nonhuman primate cynomolgus ES cells into fetal sheep at 43-67 gestational days (full term 147 days, n=15). After birth, cynomolgus tissues, which were mature teratomas, had been engrafted in sheep when more than 1 x 10(6) ES cells were transplanted at <50 gestational days. Despite the sustained engraftment, both cellular and humoral immune responses against the ES cells were detected, and additional transplantation was not successful in the animals. At 2 weeks post-transplantation, the ES cell progeny proliferated when transplanted at 48 (<50) gestational days, whereas they were cleared away when transplanted at 60 (>50) gestational days. These results support the rapid development of the xenogeneic immunological barrier in fetal sheep after 50 gestational days. Notably, a large number of Foxp3(+) regulatory T cells were present around the ES cell progeny, but macrophages were absent when the transplant was conducted at <50 gestational days, implying that regulatory T cells and premature innate immunity might have contributed to the sustained engraftment. In conclusion, long-term macroscopic engraftment of primate ES cells in sheep is feasible despite the xenogeneic immunological barrier.

Histochemical properties of bovine and ovine mammary glands during fetal development.

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Duodenal infusion of fatty acids differentially affects plasma glucagon-like peptide-1 and ghrelin concentrations in sheep.

The aim of this study was to investigate how intraduodenal infusions of fatty acids (FA) affect appetite-related gut peptides such as glucagon-like peptide-1 (GLP-1) and ghrelin in sheep. We hypothesized that these peptides can be highly reactive to unsaturated long-chain FA, because they are well known to decrease dry matter intake (DMI). Four ewes were fitted with a duodenal cannula and a jugular vein catheter for a 6-h duodenal infusion of the 9 FA (C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, and C18:3) and water (control). The concentration of each FA was 1.6 g per metabolic body weight (BW), approximately corresponding to the amount of supplemented fat in a standard dairy cow diet. Each infusion was separated by at least 2 d. During the infusion period, blood samples were collected periodically to determine changes in plasma GLP-1, ghrelin, and metabolite concentrations. Duodenal infusions of C18:1, C18:2, and C18:3 led to higher plasma GLP-1 (P < 0.05) and lower glucose (P < 0.05) than control. Plasma ghrelin concentrations were greater in C18:1 and C18:3 infusions than control (P < 0.05). Plasma ketone bodies were higher in C8:0 and C10:0 infusions (P < 0.05), but plasma triglyceride concentrations were lower in C8:0, C10:0, C12:0, and C16:0 infusions (P < 0.05) than control. Fatty acid infusions except for C18:3 led to higher plasma NEFA concentrations than control (P < 0.05). These results confirmed that the hypophagic effect of dietary unsaturated long-chain FA is mediated by GLP-1 (an anorexigenic effect) secretion. However, we also observed higher plasma ghrelin (an orexigenic effect) partially by unsaturated long-chain FA. Thus, the gut peptide secretions when ruminant animals ingest FA supplements would complexly affect satiety and further studies are needed to determine their each impact on DMI.